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Laboratory Information Bulletin No. 4306
June 2003
Denver District Laboratory and
Animal Drugs Research Center
Food and Drug Administration
P.O. Box 25087, Denver CO 80225-0087
The Laboratory Information Bulletin is a tool for the rapid dissemination of laboratory methods (or information) which appear to work. It may not report completed scientific work. The user must assure him/herself by appropriate validation procedures that LIB methods and techniques are reliable and accurate for his/her intended use. Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
This LIB describes a solid phase extraction and cleanup procedure for the determination of chloramphenicol residues in shrimp and crab. Chloramphenicol is detected, after chromatographic separation, using a Thermo Electron TSQ Quantum triple quadrupole mass spectrometer with an ESI source operating in negative ion mode. An internal standard is used to correct for variability of extraction and instrument response. Four product ions of chloramphenicol and one product ion of the internal standard are monitored using selected reaction monitoring (SRM). Chloramphenicol can be quantitated and confirmed at the 0.1ppb level.
Recently it has been reported that the antibiotic chloramphenicol (CAP) has been found in several imported foodstuffs from Asia, including shrimp, crab and crayfish. Initially, several confirmatory analytical LC/MS/MS methods for chloramphenicol using ion trap detection were developed by the Denver District Animal Drugs Research Center (ADRC) of FDA to address this problem. Specifically, LIBs 4284, 4287, 4294, 4281 (1-4) describe methods developed to qualitatively confirm CAP in shrimp, crayfish, crab, and honey, respectively, at 1ppb or higher. Using these methods over the last year, several FDA laboratories have positively confirmed the presence of CAP in many import samples of the aforementioned foods. These methods employ negative ion mode with electrospray ionization. In these methods full scan MS2 spectra were obtained not only from the parent CAP ion (m/z=321), but also from the corresponding m/z =323 ion M+2 (35Cl, 37Cl) isotope. The ion trap full scan data gave excellent positive identification but was not quite as successful for precise quantitation or sensitive enough to detect sub-ppb CAP levels. It was requested that a more sensitive confirmatory method for CAP be developed for use in the field.
To gain this added sensitivity, typically a triple quadrupole LC/MS/MS operating in Selected Reaction Monitoring or SRM mode is used. In SRM, instead of monitoring the entire mass spectrum of the product ions of a parent ion, only a few selected product ions are monitored. Seattle District Laboratory has recently developed a triple quadrupole Finnigan TSQ 7000 LC/MS/MS method (5) for the analysis of CAP in shrimp (LIB 4290) which seems to work well with a LOQ of 0.3ppb and a LOD of 0.08ppb. However, our newer model triple quadrupole instrument (a TSQ Quantum, also using negative ion mode ESI LC/MS/MS) gave appreciable downward drift in response to chloramphenicol after many injections during a run sequence, possibly due to ion suppression. Another FDA laboratory (verbal communication) has also experienced a similar effect operating in negative ion mode. For this reason we would not be able to use the Seattle method without an internal standard.
This LIB describes an additional method for the LC/MS/MS detection of chloramphenicol in shrimp and crab. Differences between our method and the Seattle method include:
An internal standard (IS), meta-Chloramphenicol or m-CAP, is added at the beginning of the extraction in our method. The use of an internal standard self-corrects for any extraction variability from sample to sample and should also self -correct for any drift in detector response during a run. With the use of an IS, 0.1ppb CAP levels in tissue can be reliably quantitated.
The extraction employed in our method does not use separatory funnels but uses disposable glassware to minimize the possibility of cross-contamination.
A mobile phase of only acetonitrile and water without any salt buffers is used in our method which should help minimize MS maintenance.
The extraction used in the method described in this LIB is a simplified version of that described in LIB 4284. Ten grams of tissue composite (with added m-CAP internal standard) is extracted (3x) in ammoniated ethyl acetate. The combined extracts are evaporated and re-dissolved in water. A hexane wash removes fats. The extract is then applied to a reverse-phase SPE column and the CAP is eluted with methanol. The methanol is evaporated to dryness and the extract is reconstituted in water, filtered and injected into the LC/MS for analysis. The m/z 321 [M-H] ion for both CAP and the internal standard m-CAP is isolated in Q1, with five product ions isolated in Q3. Confirmation of CAP is made by comparison of ion chromatogram peaks with those of a CAP standard. Quantitation of CAP is made by taking the ratio of the CAP base peak area (m/z 152) to the internal standard m-CAP base peak area (m/z 207). This MS approach is an adaptation (as per a manuscript kindly provided to Denver Laboratory by FDA's Center for Veterinary Medicine) of a Canadian method (6). The Canadian method's HPLC column with a modified mobile phase is used in this LIB. Also we found that by adding the internal standard m-CAP at the beginning of the extraction instead of at the end increased the reproducibility of the method. Finally, we also used a higher level of internal standard (0.3ppb) to give a more intense response.
The internal standard used is m-chloramphenicol (m-CAP), was custom synthesized for the FDA.
Chloramphenicol standard used is the current USP lot.
MS Editor Page (Segments & Scan Events) note: CE=collision energy, scan time in sec
Number of Scan Events: 5
Parent | Product | Width | Time | CE | Q1 PW | Q3 PW | Tube Lens |
---|---|---|---|---|---|---|---|
320.900 | 152.000 | 1.500 | 0.20 | 22 | 0.90 | 0.90 | Tune Value |
Parent | Product | Width | Time | CE | Q1 PW | Q3 PW | Tube Lens |
---|---|---|---|---|---|---|---|
320.900 | 176.000 | 1.500 | 0.20 | 20 | 0.90 | 0.90 | Tune Value |
Parent | Product | Width | Time | CE | Q1 PW | Q3 PW | Tube Lens |
---|---|---|---|---|---|---|---|
320.900 | 194.000 | 1.500 | 0.20 | 18 | 0.90 | 0.90 | Tune Value |
Parent | Product | Width | Time | CE | Q1 PW | Q3 PW | Tube Lens |
---|---|---|---|---|---|---|---|
320.900 | 257.000 | 1.500 | 0.20 | 17 | 0.90 | 0.90 | Tune Value |
Parent | Product | Width | Time | CE | Q1 PW | Q3 PW | Tube Lens |
---|---|---|---|---|---|---|---|
320.900 | 207.000 | 1.500 | 0.20 | 18 | 0.90 | 0.90 | Tune Value |
Divert Valve Page number of valve positions = 3
Divert Time (min) | Valve State |
---|---|
0.00 | Inject \ Waste |
4.00 | Load \ Detector |
5.50 | Inject \ Waste |
General:
Solvent A name: Water
Solvent B name:
Solvent C name:
Solvent D name: Acetonitrile
Column name:
Min. Pressure, bar: 10
Max. Pressure, bar: 400
Pumping Efficiency, %: 100
Fractionations/Filling Stroke: 1
Use custom stability limits: No
No. | Time, min | Flow, ul/min | A, % | B, % | C, % | D, % | |
---|---|---|---|---|---|---|---|
1 | 0.00 | 200 | 65 | 0 | 0 | 35 | |
2 | 6.00 | 200 | 65 | 0 | 0 | 35 | |
3 | 6.50 | 200 | 10 | 0 | 0 | 90 | |
4 | 13.50 | 200 | 10 | 0 | 0 | 90 | |
5 | 14.00 | 200 | 65 | 0 | 0 | 35 | |
6 | 20.00 | 200 | 65 | 0 | 0 | 35 |
The [M-H-] (m/z321) ion for both CAP and the internal standard m-CAP is isolated
(after LC separation) in Q1 with five product ions isolated in Q3.
Specifically:
Product ion m/z | Ion |
---|---|
CAP | |
257 | [M-CO, HCl]- |
194 | [M-H- Cl2HCCONH2]- |
176 | [194-water]- |
152 | [O2N-C6H4-CHOH]- (used as CAP base peak) |
m-CAP | |
257 | [M-CO, HCl]- |
207 | [257-CH3Cl and rearrangement]- (m-CAP base peak) |
For positive CAP confirmation, the CAP MS2 ions of 152, 176, 194, and 257 should all be present at the same retention time (within 5%) as that of standard CAP. These ions should each be present with area abundances of at least one-half the abundance of the 0.1ppb CAP spike. The m-CAP should be present in all samples and the 207 m-CAP SRM should be present at the same retention time (within 5%) as compared to that of standard m-cap for positive confirmation of the internal standard.
Using the collision energies as specified in the instrument SRM table, the CAP MS2 product ions have the following average peak area ratios for multiple injections of CAP standards (normalized to the 152 ion as 100%).
m/z 257 |
m/z 194 |
m/z 176 |
---|---|---|
48% | 20% | 23% |
As part of the confirmation criteria for CAP, any presumptive CAP tissue positive must have similar ion abundance ratios to the average of the CAP standards (within 10% absolute difference). CAP tissue spikes in our laboratory typically gave % RSD's of <5% for ion peak area ratios.
For CAP quantitation, use m/z 321>152 peak area for CAP response and m/z 321>207 peak area for m-CAP response. Construct a calibration curve using all standards (including the zero standard) using the ratio of [(CAP 152)/(m-CAP 207)] responses vs. CAP concentration in ng/mL. Determine the concentration of the sample extract from the calibration curve. Calculate the corresponding tissue concentration by: (assuming an initial 10g tissue portion, and a final vial (extract) volume of 0.5mL)
[ng/g x (10g/0.5mL)]=ng/mL or [ ppb x 20] = ng/mL, or
Tissue ppb=[extract conc. in ng/mL]/20
The MS response to chloramphenicol was found to have a limited linear range. To quantitate at levels around 0.1ppb, a linear standard curve from 0 to 1ppb can be used (such curves gave correlation coefficients (r) of >0.995). However, to quantitate at both low and high (>1ppb CAP) levels a quadratic fit curve over a range of 0 to 5ppb was sometimes found to work better. Although a linear fit curve from 0-5ppb CAP might still have a r value of >0.995, using this curve often would adversely affect quantitation at low levels of 0.1ppb. (See figs 4 and 5).
Using an internal standard, repeat injections of a CAP standard (or CCV standard) should easily give values of <25% difference from the initial value. Similarly, an injection of an independently made CAP standard (or ICV standard) should also easily give an agreement within 25% of the CCV CAP standard. A 0.1ppb CAP standard (2ng/mL CAP) gave an average signal-to-noise ratio (S/N) of 800:1 for SRM 152 and an average S/N ratio of 1400:1 for SRM 207 (m-CAP internal standard at 6ng/mL or 0.3ppb m-CAP-see Fig. 1).
Quantitation of 0.1ppb CAP levels allows the 3:1 compositing of individual tissue subs to be done and still ensures that a 0.3ppb quantitation level be maintained in any individual sub. (A 3-sub composite could have one sub containing 0.3ppb CAP blended (composited) with two subs containing no chloramphenicol and the composite would still be confirmed for CAP). See table 1 for recovery data of chloramphenicol in shrimp and crab.
Figure 1 : 0.1ppb or 2ng/mL CAP std with 6ng/mL internal standard m-CAP
Plot of MS2 ions m/z 152, 176, 194, 257 and 207 (m-CAP).
Figure 2: 0.1ppb CAP shrimp spike with internal standard m-CAP spiked at 0.3ppb (equiv. to 6ng/mL m-CAP)
Figure 3: Control shrimp blank spiked with 0.3ppb internal standard m-CAP
Figure 4. CAP standard curve from 0-3ppb CAP (0-60ng/mL) showing quadratic fit
Cap concentration in ng/mL |
Figure 5. CAP standard curve over smaller range of 0-1ppb CAP showing linear fit
Cap concentration in ng/mL |
Spike level (ppb) | Number of spikes (n) | Tissue matrix | Avg. % recovery | RSD (%) |
---|---|---|---|---|
0.05 | 3 | Shrimp | 87 | 4 |
0.1 | 11 | Shrimp | 89 | 25 |
0.2 | 3 | Shrimp | 99 | 16 |
0.3 | 12 | Shrimp | 101 | 14 |
0.6 | 7 | Shrimp | 95 | 9 |
2 | 1 | Shrimp | 95 | na |
0.05 | 3 | Crab | 76 | 4 |
0.1 | 5 | Crab | 106 | 23 |
0.2 | 6 | Crab | 100 | 28 |
0.3 | 6 | Crab | 101 | 17 |
1 | 3 | Crab | 92 | 6 |
2 | 3 | Crab | 97 | 2 |
A. Pfenning, S. Turnipseed, J. Roybal, C, Burns, M. Madson, J. Storey and R. Lee, "Confirmation of Multiple Phenicol Residues in Shrimp by Electrospray LC/MS" (2002) Laboratory Information Bulletin 4284 13 pages.
S. Turnipseed, C, Burns, J. Storey, R. Lee, and A. Pfenning, "Confirmation of Multiple Phenicol Residues in Honey by Electrospray LC/MS" (2002) Laboratory Information Bulletin 4281 12 pages.
A. Pfenning, S. Turnipseed, J. Roybal, C, Burns, M. Madson, J. Storey and R. Lee, "Confirmation of Multiple Phenicol Residues in Crab by Electrospray LC/MS" (2002) Laboratory Information Bulletin 4294 12 pages.
A. Pfenning, S. Turnipseed, J. Roybal, C. Burns, M. Madson, J. Storey and R. Lee, "Confirmation of Multiple Phenicol Residues in Crawfish by Electrospray LC/MS" (2002) Laboratory Information Bulletin 4289 12 pages.
B. Neuhaus, J. Hurlbut, and W. Hammack, "LC/MS/MS Analysis of Chloramphenicol in Shrimp" (2003) Laboratory Information Bulletin 4290 18 pages
Quon, et. al, "Chloramphenicol Residues in Honey", (2002), Canadian Food Inspection Agency, Calgary Food Laboratory, Additives and Chemical Contaminants Analytical Methods Manual, Method ACC-062-V1.0, Volume 5.
E. Bunch, D. Altwein, L. Johnson, J. Farley and A. Hammersmith, (1995), J. AOAC Int. 78 (3) 883-887.